Fura 2 filter

Calcium levels were live monitored in attached astrocytes through the ratiometric probe Fura-2-AM (#F1221, Invitrogen) as done by Kowaltowski et al. (2019) (link). Briefly, cells were plated in glass-bottom culture dishes (#627871, Greiner Bio-One), incubated with 5 μM Fura-2-AM for 30 min at 37°C in experimental medium lacking FBS and supplemented with 1 mg/ml bovine serum albumin (BSA). Fluorescence was assessed at λ_ex = 340 (F₃₄₀) and 380 nm (F₃₈₀) and λ_em = 510 nm in a Leica DMi-8 microscope equipped with a Fura-2 filter (Leica Microsystems). Cells were followed through additions of CGP (10 μM), ATP (100 μM), as well as ionomycin (20 μM) to allow calibration. Analyses were conducted through FIJI ImageJ 1.52p (Schindelin et al., 2012 (link)), in which individual cells (55–125/group per experiment) were identified as regions of interest (ROI) and [Ca²⁺] variation was estimated as the ratio (R) between F₃₄₀/F₃₈₀. Data were calibrated by the maximal ratio induced by ionomycin, controlled for background fluorescence oscillations, and normalized by the initial ratio (R₀).